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Cx43 Regulated The ERK1/2 Phosphorylation Involved In Nicotine-induced Mice Distal Pulmonary Artery Remodeling

Posted on:2024-07-05Degree:MasterType:Thesis
Country:ChinaCandidate:X R XuFull Text:PDF
GTID:2544307148977159Subject:Public health
Abstract/Summary:PDF Full Text Request
Objective:To study the mechanism of the regulation of ERK1/2 phosphorylation by Cx43 on the remodelling of distal pulmonary artery induced by nicotine in mice at the animal and cellular levels,and to provide a theoretical basis for the search for a new target for prevention and treatment of pulmonary hypertension.Methods:Part Ⅰ:1.Using CRISPR/Cas9 method,Cx43 gene mice were hybridized with vascular smooth muscle specific tool mice,and Tagln-Cre and Myhl 1-Cre/ERT2 vascular smooth muscle specific knockout mice with Cx43 gene were established.There were 32 male C57BL/6J mice and 32 male Tagln-Cre(+);Cx43flox/WT mice were randomly divided into Saline group,0.02 mg/kg/d Nicotine group,0.2 mg/kg/d Nicotine group and 2 mg/kg/d Nicotine group,respectively.Mice in each group received Nicotine nasal drops for eight weeks and Nicotine-poisoned pulmonary artery remodeling model was established.2.The general condition and body weight changes of mice were observed and survival analysis was performed.The pulmonary artery histological changes were observed by HE staining and WA%was measured.The expressions of α-SMA and Cx43 in mice distal pulmonary artery were observed by immunofluorescence staining.Part Ⅱ:1.The mice primary distal PASMCs were cultured by magnetic separation.The mice primary distal PASMCs were treated with nicotine in different concentrations(10-8 M,10-7 M,10-6 M,10-5 M,10-4 M,10-3 M)for different times(6 h,12 h,24 h,48 h),and the cell activity was determined by CCK-8 assay.Nicotine in different concentrations(10-6 M,10-5 M,10-4 M)was applied to the mice primary distal PASMCs for 24 h.The proliferation of the mice primary distal PASMCs was determined by EdU assay,the cell cycle change of the mice primary distal PASMCs was determined by flow cytometry,and the migration ability of the mice primary distal PASMCs was determined by wound-healing assay.The expressions of PCNA in mice distal pulmonary artery were observed by immunofluorescence staining.2.The primary distal PASMCs of C57BL/6J mice and Tagln-Cre(+);Cx43flox/WT mice were divided into Control-PASMCs group,Cx43flox/WT-PASMCs group,ControlPASMCs+Nicotine group,Cx43flox/WT-PASMCs+Nicotine group,Ctrl-PASMCs+PD98059 group,and Control-PASMCs+PD98059+Nicotine group.Nicotine in different concentrations(10-6 M,10-5 M,10-4 M)was applied to the mice primary distal PASMCs for different times(12 h,24 h,48 h,72 h).Western blot was used to detect the expression of Cx43,p-ERK1/2 and ERK1/2.Cx43flox/WT-PASMCs and PD98059 were used to inhibit the expression of Cx43 and p-ERK1/2,respectively.The mice primary distal PASMCs of each group were exposed to 10-5 M nicotine for 24 h.The expressions of Cx43,p-ERK1/2 and ERK1/2 were detected by Western blot.Cell proliferation was observed by EdU assay.Results:Part I:1.C57BL/6J mice of Nicotine-poisoned pulmonary artery remodeling model had no significant difference in body weight before poisoning,and the higher the Nicotine dose,the slower weight gain.Compared with Saline group,the body weight of C57BL/6J mice in 0.2 mg/kg/d and 2 mg/kg/d Nicotine groups decreased significantly from the 6th and 4th week after Nicotine poisoning,respectively(P<0.05).The survival analysis showed that the survival rate of mice in 2 mg/kg/d Nicotine group was lower than that in Saline group,0.02 mg/kg/d and 0.2 mg/kg/d Nicotine group,with statistical difference(P<0.05).2.Nicotine promoted the upregulation of Cx43 in distal pulmonary artery of C57BL/6J mice.Immunofluorescence results of lung tissues showed that Cx43 expression in mice distal pulmonary artery of all groups significantly decreased compared with C57BL/6J group(P<0.001).Compared with Myh11-Cre/ERT2 vascular smooth muscle specific knockout mice with Cx43 gene,the efficiency of Cx43 knockout in distal pulmonary artery of Tagln-Cre mice was significantly reduced(P<0.001).Tagln-Cre(+);Cx43flox/flox mice showed homozygous lethality,so Tagln-Cre(+);Cx43flox/WT mice were used as the follow-up experimental vascular smooth muscle specific knockout mice with Cx43 gene in this study.As the dose of Nicotine increased,compared with Saline group,the expression of Cx43 in the distal pulmonary artery of C57BL/6J mice in each Nicotine groups significantly increased(P<0.001).The fluorescence intensity of Cx43 in the distal pulmonary artery of Tagln-Cre(+);Cx43flox/WT mice poisoned with Nicotine in each dose was weaker than that of C57BL/6J mice(P<0.001).3.Down-regulation of Cx43 alleviated nicotine-induced pulmonary artery remodeling in mice.As the Nicotine dose increased,the remodeling degree of distal pulmonary artery in C57BL/6J mice increased gradually.Compared with Saline group,the degree of C57BL/6J mice distal pulmonary artery muscularization of Nicotine poisoned groups increased significantly in a dose-dependent manner(P<0.001).The results of HE staining showed that the wall of the distal pulmonary artery of C57BL/6J mice in Nicotine poisoned groups gradually thickened,the lumen narrowed with the increase of Nicotine dose and WA%increased in a dose-dependent manner,with infiltration of lymphocytes,neutrophils and mononuclear macrophages,and 2 mg/kg/d Nicotine group was the most significant.Compared with Saline group,WA%of C57BL/6J mice in 0.2 mg/kg/d and 2 mg/kg/d Nicotine groups increased significantly in the distal pulmonary duct wall(P<0.001).Except Saline group,distal pulmonary artery muscularization of Tagln-Cre(+);Cx43flox/WT mice was weaker than that of C57BL/6J mice in each dose(P<0.001).The results of HE staining showed that the duct wall of Tagln-Cre(+);Cx43flox/WT mice in Nicotine poisoned groups had no significant change,no inflammatory cell infiltration and intact alveolar structure.Compared with Saline group,there was no significant difference in WA%of Tagln-Cre(+);Cx43flox/WT mice in Nicotine poisoned groups(P>0.05).In addition,the WA%of Tagln-Cre(+);Cx43flox/WT mice distal pulmonary duct wall decreased significantly in the concentration of 0.2 mg/kg/d and 2 mg/kg/d Nicotine groups compared with C57BL/6J mice(P<0.001).These results suggested that the knockout of Cx43 could reverse the mice distal pulmonary remodeling induced by nicotine.Part Ⅱ:1.Niotine poisoning could promote the viability,proliferation,cell cycle conversion,and migration of the mice primary distal PASMCs in a dose-dependent manner.The typical "long spindle" morphology and α-SMA immunofluorescence results showed that the mice primary distal PASMCs could be obtained by magnetic separation.The results of CCK-8 showed that there was no significant difference in the cell viability of the mice primary distal PASMCs in C57BL/6J mice before poisoning(P>0.05).In all time groups,the cell activity of primary distal PASMCs significantly increased after 10-8 M-10-4 M nicotine poisoning,and the promotion effect of 10-5 M nicotine was the most significant.While 10-3 M Nicotine significantly inhibited cell viability at 6 h(P<0.001)and 12 h(P<0.05).The cell viability of the mice primary distal PASMCs exposured to Nicotine at 10-7 M to 10-3 M significantly increased in each time group compared with 6 h,and 24 h was the most significant effect.The EdU results showed that 10-6 M,10-5 M,and 10-4 M Nicotine could significantly promote the proliferation of primary distal PASMCs in C57BL/6J mice compared with the Control group(P<0.001).In addition,10-5 M Nicotine promoted the proliferation of C57BL/6J mice primary distal PASMCs most significantly(P<0.001).Flow cytometry showed that the S phase of the C57BL/6J mice primary distal PASMCs treated with 10-5 M and 10-4 M Nicotine significantly increased(P<0.001)and G1 phase significantly decreased(P<0.001)compared with the Control group.The results indicated that Nicotine could promote the G1 to S transformation of distal PASMCs cell cycle in C57BL/6J mice,and 10-5 M Nicotine had the most significant effect.Wound-healing results showed that the PASMCs migration ability of C57BL/6J mice primary distal PASMCs treated with 10-5 M and 10-4 M Nicotine significantly increased compared with the Control group(P<0.001),and the ability of 10-5 M Nicotine to promote primary distal PASMCs migration in C57BL/6J mice was the most significant(P<0.001).As the Nicotine dose increased,PCNA expression in the distal pulmonary artery of C57BL/6J mice in each Nicotine group significantly increased(P<0.001)compared with saline group,as well as 2 mg/kg/d Nicotine group(P<0.001).2.Nicotine promoted the up-regulation of Cx43 expression in the primary distal PASMCs of C57BL/6J mice.Compared with the Control group,the relative expression level of Cx43 protein in C57BL/6J mice primary distal PASMCs poisoned 10-5 M and 10-4 M Nicotine significantly increased(P<0.05).Compared with 0 h group,the relative expression level of Cx43 protein in 24 h group significantly increased(P<0.001).Based on the above results,the mice primary distal PASMCs were treated with 10-5 M Nicotine for 24 h as the follow-up experiment condition.3.The up-regulation of Cx43 expression promoted the phosphorylation of ERK1/2 and participated in the proliferation of primary distal PASMCs induced by nicotine.Cell immunofluorescence results showed that compared with C57BL/6J group,the expression of Cx43 in the mice primary distal PASMCs of all groups significantly decreased(P<0.001),and the knockout efficiency of Cx43 in primary distal PASMCs of Tagln-Cre(+);Cx43flox/WT mice was higher(P<0.001).Therefore,primary distal PASMCs of Tagln-Cre(+);Cx43flox/WT mice were adopted as the primary distal PASMCs of vascular smooth muscle specific knockout mice with Cx43 gene in the follow-up experiments of this study.Compared with the Control group,the relative expression level of p-ERK1/2 protein in the primary distal PASMCs of C57BL/6J mice poisoned by 10-5 M Nicotine the most significant increased(P<0.05);compared with 0 h group,the relative expression level of p-ERK1/2 protein in 12 h group and 24 h group significantly increased(P<0.001),and there was no significant difference in the relative expression level of ERK1/2 protein among all groups,suggesting that nicotine could promote phosphorylation of ERK1/2 protein in the C57BL/6J mice primary distal PASMCs.Compared with Ctrl-PASMCs group,the relative expression level of p-ERK1/2 protein in Cx43flox/WT-PASMCs group significantly decreased(P<0.05);compared with Ctrl-PASMCs+Nicotine group,the relative expression level of p-ERK1/2 in Cx43flox/WT-PASMCs+Nicotine significantly decreased(P<0.001).There was no significant difference in the relative expression of ERK1/2 protein among all groups.It is suggested that Cx43 knockout can inhibit ERK1/2 phosphorylation.The cell proliferation of Ctrl-PASMCs+Nicotine group significantly increased compared with Ctrl-PASMCs group(P<0.001).Compared with Ctrl-PASMCs+Nicotine group,the cell proliferation of Cx43flox/WT-PASMCs+Nicotine group significantly decreased,suggesting that Cx43 down-regulation could significantly inhibit the proliferation of the mice primary distal PASMCs(P<0.001).The cell proliferation of Ctrl-PASMCs+Nicotine group significantly increased compared with Ctrl-PASMCs group(P<0.001).Compared with Ctrl-PASMCs+Nicotine group,the proliferation of cells significantly decreased in the Ctrl-PASMCs+PD98059+Nicotine group,suggesting that ERK1/2 inhibition could significantly inhibit the proliferation of the mice primary distal PASMCs(P<0.001).These results suggested that up-regulation of Cx43 contributed to the phosphorylation of ERK1/2 protein in the proliferation and migration of the mice primary distal PASMCs induced by Nicotine.Conclusions:1.Nicotine-induced distal pulmonary artery remodeling in mice was related to the up-regulation of Cx43 expression,and the specific knocking out of Cx43 in vascular smooth muscle could significantly alleviate nicotine-induced pulmonary artery remodeling in mice.2.Nicotine could induce the proliferation and migration of primary distal PASMCs in mice,and its mechanism may be related to the up-regulation of Cx43 expression of distal PASMCs,thus promoting the phosphorylation of ERK1/2.
Keywords/Search Tags:Gap junction Connexin43, Pulmonary vascular remodeling, Cell proliferation and migration, nicotine
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